Original ArticlesCharacterization of gastrin-releasing peptide receptors aberrantly expressed by non-antral gastric adenocarcinomas
Introduction
Bombesin is a tetradecapeptide originally isolated from the skin of the frog Bombina bombina [8]. The mammalian homolog of bombesin, gastrin-releasing peptide (GRP) binds to and activates a specific 7 transmembrane-spanning G protein coupled (heptaspanning) receptor [1], [6], [33]. In non-malignant tissues, GRP receptor (GRP-R) activation causes numerous diverse effects in the central nervous, immune, pulmonary, and gastrointestinal (GI) systems (reviewed in [34]). In the stomach, GRP-R are normally expressed by G cells resident in the mucosal epithelium lining the antrum where they are involved in the regulation of acid secretion [35]. Although GRP-R are not normally expressed by mucosal epithelial cells lining the human GI tract outside of the gastric antrum [10], the precise region of the stomach normally expressing this receptor is not known.
Recent studies indicate that many GI cancers and cancer cell lines aberrantly express GPR-R including those derived from the stomach [14], [24], [27], pancreas [26], and colon [12], [13], [29], [30]. In all studies GRP-R antagonists attenuate the growth of these GI cancer cell lines [14], [26], [27], [29], [30], leading to a presumption that this receptor significantly contributes to tumor growth and adversely affects patient prognosis. We were initially interested in determining whether these aberrantly expressed receptors could be mutated in such a way as to contribute to tumor cell growth, because earlier studies had revealed significant differences in their pharmacology as compared to GRP-R expressed by non-GI tissues (reviewed in [10]). Based on our prior work demonstrating that alterations in receptor-G protein coupling could alter GRP-R pharmacology [2], [11], [16], we hypothesized that these aberrantly expressed receptors were mutated causing them to become constitutively active. Such a mechanism would provide a novel mechanism whereby the GRP-R could stimulate tumor cell growth and adversely alter patient outcome.
Heptaspanning receptor mutation is increasingly accepted as the cause for a number of diseases (reviewed in [15]). The only mutated heptaspanning receptor associated with cancer is the thyroid-stimulating hormone receptor (TSH-R). Recent studies indicate that many thyroid adenomas and tumors express mutated TSH-R that are constitutively active such that they can activate second messengers independently of ligand (reviewed in [23]). Given the increasing frequency with which it is appreciated that heptaspanning receptor mutations can cause various human diseases, and given the altered pharmacology of GRP-R aberrantly expressed in GI cancers, we set out to study this receptor in adenocarcinomas of the stomach. To restrict our evaluation to tumors aberrantly expressing this receptor, we first determined the limits of normal GRP-R expression by mucosal epithelial cells lining the stomach. We then evaluated the RNA previously extracted from 20 consecutive gastric tumors located outside the region not normally expressing this receptor. We herein report that aberrant GRP-R expression by non-antral gastric adenocarcinomas is common, that these receptors are frequently mutated, with mutations resulting in non-functional as well as constitutively active receptors. Unexpectedly, we also found that aberrant expression of functional GRP-R is not associated with a decrease in patient survival. This suggests the possibility that GRP-R in at least gastric cancer may not act as a clinically significant growth factor.
Section snippets
Materials
All radionucleotides were obtained from Amersham (Arlington Heights, IL). Taq polymerase was obtained from Perkin Elmer (Foster City, CA), Pfu polymerase from Stratagene (La Jolla, CA), whereas murine Maloney leukemia virus reverse transcriptase and Cleavase were from Gibco/BRL (Gaithersburg, MD); all restriction enzymes were from Promega (Madison, WI). Cloning vectors including pCR2.1 and pcDNA-3 were from Invitrogen (Carlsbad, CA), whereas gel drying paper (3 mm) was from Whatman (Maidstone,
Expression of GRP-R by normal gastric epithelium
The extent of normal GRP-R expression in gastric mucosa was defined by performing serial mucosal biopsies on volunteers. We previously have shown that within the GI tract, mucosal expression of the GRP-R is confined to the gastric antrum [10]. To more precisely define the extent of normal GRP-R expression by gastric mucosal epithelium, we subjected volunteers to endoscopic biopsy every 2 cm starting at the pylorus and proceeding orad. Nested PCR on the RNA extracted from the endoscopic pinch
Discussion
This study is the first to show that non-antral gastric adenocarcinomas aberrantly express GRP-R that are frequently mutated, and that expression of functional receptor is not associated with a decrease in patient survival. Epithelial cells lining the human GI tract do not normally express GRP-R except in the distal stomach [10]. In contrast, multiple human gastric cancer cell lines [14], [24], [27], and up to 56% of surgically resected gastric cancers [25], have been found to express this
Acknowledgments
This work was supported by an ADHF/Astra Merck Advanced Research Fellowship (to R.E.Carroll); and by an ADHF/American Gastroenterological Association Industry Research Scholar Award, NIH grant DK51168, and a VA Merit Review (to R.V. Benya).
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